Antibodies to factor VIII are present in about 15% of treated patients with hemophilia A and occur as autoantibodies rarely in previously normal people. Antibodies to von Willebrand factor (vWF) have been reported in several patients with severe von Willebrand disease (vWD) and as autoantibodies in normal people on rare occasions. We are studying the factor VIII autoantibodies which developed in two previously normal people in order to determine the epitope specificities. Factor VIII was digested with either thrombin or trypsin and the proteolytic fragments immunoprecipitated with patient antibody coupled to protein A-sepharose beads. The precipitated fragments were detected by immunoblotting or by autoradiography of 125I-labelled factor VIII. These experiments demonstrated that one antibody, a high-titer, type 1 inhibitor recognized epitopes on the A1, A2, and light chain fragments whereas the other, a type 2 inhibitor, recognized the A2 region and the light chain. Binding to the A2 region was weak. Both antibodies precipitated recombinant 35S-labelled C2 region fragments. We have also studied an autoantibody to vWF. 125I- labelled vWF fragments generated by digestion with Staphylococcal V8 protease (the major principle fragments generated by S V8 are 170 kDa and 100 kDa) were immunoprecipitated by patient IgG bound to staphylococcal protein A-sepharose beads. The precipitated fragments were separated by PAGE and the proteins detected by autoradiography. The majority of the radioactivity was located in a band of molecular weight of about 170 kDa, indicating that this fragment contains the epitope recognized by the patient's antibody. Studies are in progress to further localize this epitope.